Antibiotic resistance genes in the gut microbiota of mothers and linked neonates with or without sepsis from low- and middle-income countries

Early development of the microbiome has been shown to affect general health and physical development of the infant and, although some studies have been undertaken in high-income countries, there are few studies from low- and middle-income countries. As part of the BARNARDS study, we examined the rectal microbiota of 2,931 neonates (term used up to 60 d) with clinical signs of sepsis and of 15,217 mothers screening for blaCTX-M-15, blaNDM, blaKPC and blaOXA-48-like genes, which were detected in 56.1%, 18.5%, 0% and 4.1% of neonates’ rectal swabs and 47.1%, 4.6%, 0% and 1.6% of mothers’ rectal swabs, respectively. Carbapenemase-positive bacteria were identified by MALDI-TOF MS and showed a high diversity of bacterial species (57 distinct species/genera) which exhibited resistance to most of the antibiotics tested. Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae/E. cloacae complex, the most commonly found isolates, were subjected to whole-genome sequencing analysis and revealed close relationships between isolates from different samples, suggesting transmission of bacteria between neonates, and between neonates and mothers. Associations between the carriage of antimicrobial resistance genes (ARGs) and healthcare/environmental factors were identified, and the presence of ARGs was a predictor of neonatal sepsis and adverse birth outcomes.


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Nature Microbiology and cost 6 , perturbs the gut microbiome and can modulate bacterial populations that have a negative impact on neonatal development. Gibson et al. and other studies from Dantas's group, primarily from high-income countries, have demonstrated that antibiotic therapy in preterm infants can dramatically affect the gut microbiome [7][8][9] .
Large-scale multi-national studies using molecular methods to assess the carriage of ARGs among maternal and neonatal microbiota in LMICs are non-existent. BARNARDS is a network of 12 clinical sites across 7 LMICs in Africa and south Asia aiming to assess the incidence, prevalence, risk factors, bacterial causes and burden of AMR in neonatal sepsis (https://www.ineosoxford.ox.ac.uk/ research/barnards). The genomic characterization of BARNARDS' sepsis isolates has already been discussed 10 , as well as their resistance profiles to β-lactam and aminoglycoside antibiotics, suggesting that the World Health Organization (WHO) may need to revise their antibiotic guidelines for neonatal sepsis within LMICs, where antibiotic resistance to current therapeutic recommendations is extremely high 6 .
In the present study, we characterize the Gram-negative gut microbiota of mothers and septic/non-septic neonates carrying clinically important extended-spectrum β-lactamases (ESBLs) and carbapenemase genes. We investigated statistical associations across maternal, neonatal, living environment and hospital environment domains and carriage of ESBLs and carbapenemase genes. In addition, we determined associations between neonatal/maternal carriage of ARGs, and sociodemographic and clinical environment traits. Furthermore, using whole-genome sequencing (WGS), we characterized common Gram-negative bacteria (GNBs) carrying carbapenemase genes, detailing specific variants and plasmid types across the different study sites.

Prevalence of β-lactamase genes among mothers and neonates.
Overall, BARNARDS recruited 35,040 mothers and their respective neonates (n = 36,285). In the present study, 18,148 rectal swabs were analysed to assess the presence of clinically important β-lactamases in the mothers' and neonates' gut microbiota, using the bla CTX-M-15 like gene as a marker for the presence of ESBLs and bla NDM , bla KPC and bla OXA-48 -like genes, as markers of carbapenemase genes (Fig. 1).
We found the prevalence of bla CTX-M-15 among MRs and BRs (Fig.  2ab) to be higher than previously reported [11][12][13][14][15][16][17] . The prevalence of bla OXA-48 -like genes in our African sites was similar to or lower than that of other studies [18][19][20] . The bla OXA-48 -like genes are reportedly widespread throughout south Asia 21 , but in our study this was observed only in Pakistan among BRs. It is interesting that bla NDM prevalence was higher than previously reported in Pakistan 14,22,23 , India 11,24,25 and Bangladesh 26 . Previous reports of bla NDM neonatal carriage in Africa are few and show low-frequency rates among children and pregnant women 12,20,[27][28][29] . Although bla KPC is widely disseminated throughout America and Europe, it is not common in south Asia or Africa 11,12,18,21,24,25 , as affirmed by the present study.
We analysed the neonate's age at the time of BR collection against carriage of ARGs and found that, from day 0, ARGs were consistently found among BRs (Fig. 2c), regardless of whether delivery was via caesarean section (CS) or spontaneous vaginal delivery (SVD) and whether or not neonates developed BS (Extended Data Fig. 1a-d). A steady decrease was observed for the prevalence of bla NDM (53.7% to 27.7%) and bla OXA-48 -like genes (35.4% to 0%) genes among the Asian samples through the first 14 d of life (Fig.  2c), independent of type of delivery or sepsis outcome (Extended Data Fig. 1a Bacterial diversity in maternal and neonatal gut microbiota. In total, 1,072 GNB isolates harbouring carbapenemase genes were recovered (Extended Data Fig. 2). From 412 BRs, we characterized 556 carbapenemase-positive bacteria (CPBs) comprising 33 species/ genera with 9 isolates unidentified (Extended Data Fig. 2a). K. pneumoniae (n = 161), E. coli (n = 132) and E. cloacae complex (n = 92) were most common, accounting for 69.6% (n = 378/543) and 80.6% (n = 54/67) of positive isolates for bla NDM and bla OXA-48 -like genes. K. pneumoniae and E. coli were the predominant concomitant carriers of bla NDM and bla OXA-48 -like genes (n = 46/54).
We found a wider CPB species diversity among BRs and MRs than previously described, where most were K. pneumoniae, E. coli and E. cloacae complex 14,28,29 . Evidence shows premature birth dramatically influences species richness and composition in the first months of life, enriching for E. coli, E. cloacae and Klebsiella sp. 30 . The 2,931 samples analysed in the present study were from 2,011 Articles Nature Microbiology term (69%), 736 (25%) preterm and 147 (5%) post-term neonates (1% clinical data missing).
Antibiotic resistance profiles (Extended Data Fig. 3a) were established for 298 BR and 281 MR CPBs. Resistance rates were especially high for amoxicillin (97%), imipenem and ertapenem (both 91%). The gentamicin resistance rate among BR isolates was higher (84%) than for MR isolates (68%). Although high resistance rates were expected due to our selective culture method, and isolates with intrinsic resistances were recovered 31 , these findings contrast previous findings of low AMRs in the south Asian community 32 .  (Fig. 4). The E. hormaechei STs ST113, ST171 and ST418 were dominant (Fig. 4) and E. hormaechei ST418 harbouring bla NDM-1 -like genes was recovered predominantly from neonates in BC and BK, with SNP analysis indicating genomic variability between 0 and 1,601 pairwise SNPs (Supplementary Table 1 (Fig. 5). Three PP BR ST15 K. pneumoniae isolates possessed the same ST as that of the isolate causing sepsis in the same neonate 10 . We detected 46 STs from which ST11, ST14, ST15 and ST48 were common across the collective phylogeny (Fig. 5). ST11 was predominantly found in Europe carrying bla OXA-48 or bla OXA-245 ; however, in our study n = 6/8 PP ST11 isolates carried bla NDM-7 (Extended Data Fig. 6). The bla NDM-1 was most frequent (n = 124; Fig. 5

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Risk factors for the rectal carriage of β-lactamase genes. To determine maternal, neonatal, living environment and hospital environment features associated with the carriage of bla CTX-M-15 , bla NDM or bla OXA-48 -like genes among the gut microbiota of mothers and neonates, we performed several exploratory univariate and multivariable analyses (Table 1 and Supplementary Table 2). We fitted a multivariable model including WASH (water, sanitation and hygiene)-associated features (Extended Data Fig. 7a), to understand the impact of these indicators in the carriage of the ARGs in the study among MRs. In 2017, 7% (Ethiopia) to 76% (South Africa) of the population in countries of the BARNARDS network used at least basic sanitation services (Supplementary Table 1) and we found that occasional handwashing by the mothers or households supplied with a wastewater network were independent risk factors for carrying bla CTX-M- 15 . Multivariable models showed that occasional handwashing was associated with MR carriage of bla CTX-M-15 or bla NDM , whereas frequent handwashing was associated with the carriage of bla OXA-48 -like genes (Table 1 and Supplementary Table 2a). Poor hygiene is a driver for carriage of ARGs 38 and we speculate that deficient hand hygiene, even if frequent, could be associated with the carriage of these ARGs, specially bla CTX-M-15 . We also found that a maternal infection in the 3 months before enrolment in the present study was associated with MR carriage of bla CTX-M-15 (Table 1 and  Supplementary Table 2a). Carriage of ARGs among MRs was associated with the mothers' use of antibiotics in the 3 months before enrolment (Table 1 and Supplementary Table 2a). In similar settings, previous use of antibiotics has been described as a risk factor for carriage of ESBL producers/MDR isolates 15,38 , but this was not supported from findings in other studies 18,29,39 . We did not find an association between neonates' age at time of sampling and carriage of ARGs (Supplementary Table 2b). Previously, increased neonatal age was associated with carriage of ESBL producers 13 . β-Lactamase gene carriage and birth outcomes. Our exploratory analysis suggests that planned or emergency CS or premature birth may be associated with the mother's carriage of either bla CTX-M-15 or bla NDM . Also, the odds of having preterm premature rupture of membranes (PPROM) were higher for mothers carrying bla CTX-M-15 or bla NDM , whereas the odds of having perinatal asphyxia or a breech birth were higher for mothers carrying bla CTX-M-15 (Supplementary Table 2c).
As the hospital environment has been associated with carriage of ARGs 19,29 , we investigated whether neonates born within clinical sites (birth cohort) were more likely to carry β-lactamase genes compared with those born elsewhere (admission cohort, admitted with suspected sepsis). We found that the odds of carrying bla NDM were higher for neonates from the birth cohort (Supplementary  Table 2d). We did not find significant associations between birth healthcare/environment factors and the carriage of these ARGs among the birth cohort (Extended Data Fig. 7b and Supplementary  Table 2a). Univariate analysis including neonates from both cohorts showed that those born by emergency CS were more likely to carry bla CTX-M-15 in agreement with other studies 13,39 and neonates born after PPROM had higher odds of carrying bla OXA-48 -like genes (Supplementary Table 2e).  Articles Nature Microbiology β-Lactamase gene carriage and neonatal sepsis. We found that colonization of the mother's gut with bla CTX-M-15 or bla NDM positive microbiota was associated with the development of BS in the neonate, and this may be due to the mother transmitting MDR pathogens to her neonate during or after labour and birth, potentially leading to neonatal BS 40 . Neonates carrying bla CTX-M-15 or bla OXA-48 -like genes in their microbiota were more likely to have BS compared with non-carriers (Supplementary Table 2f).

Discussion
In the present study, we report high carriage of bla CTX-M-15 , bla OXA-48 -like genes and bla NDM among the rectal microbiota of mothers and neonates with either suspected or confirmed BS. Carriage of genes was higher for neonates compared with mothers, as previously reported 15,17 , particularly, for bla NDM in samples from Bangladesh, Nigeria and Pakistan (Fig. 2). We speculate that, because most of these neonates have been administered antibiotics, if presenting with clinical sepsis, antibiotic selection pressure favoured resistant bacteria, as described previously 7,15,18,19 . We highlighted the carriage of ARGs in neonates from the very early hours after birth, irrespective of delivery type or sepsis outcome, which may have been underpinned by antibiotic therapy after acquisition from the mother and/or environment. Our results further highlight the importance of access to safe water, sanitation and good hygiene to reduce the mortality rate. WASH-related factors might have been associated with the carriage of bla CTX-M-15 among MRs, and the carriage of bla CTX-M-15 or bla NDM with poor birth outcomes and neonatal sepsis. Similarly, previous maternal infection and use of antibiotics were associated with the carriage of β-lactamase genes among MRs, and further associated with more adverse birth outcomes and neonatal sepsis. In addition, our exploratory analysis suggested that complicated births such as PPROM and clinical interventions such as a CS could be associated with neonatal ARG carriage and neonatal sepsis. We acknowledge that all statistical analyses performed are exploratory and not causal. Other uncharacterized covariates such as medical history and/or socioeconomic factors are also likely to add to the AMR burden and poor health outcomes.
The genomic analysis unveiled the existence of indistinguishable E. coli isolates from MRs and BRs, suggesting transmission from mother to neonate during or after labour. Furthermore, K. pneumoniae, which was found to be the most common cause of sepsis in neonates enrolled in BARNARDS 10 , was also the most prevalent isolate among BRs. SNP analysis revealed three cases where K. pneumoniae BRs and sepsis isolates from the same neonate were very closely related, indicating that transmission events either in the clinical setting or in the newborn gut microbiota might have occurred. In addition, SNP analysis of E. coli, E. cloacae and K. pneumoniae genomes from neonates attending the same clinical sites indicated clonal cases. Moreover, ARGs, and in particular bla NDM -like genes, were found in different plasmids (IncX3 in Bangladesh; IncN2 or IncA/C2 in Pakistan), emphasizing a diverse dissemination of MDR pathogens harbouring ARGs. The identification of ARGs in the microbiota of neonates from the first hours of life indicates ST    Articles Nature Microbiology that initial colonization occurred at birth through contact with the mother and/or the hospital environment (for example, CS), and WGS analysis shows evidence for both routes of neonatal gut colonization with AMR microbiota. These findings support the need for future studies assessing mother/non-clinical environment-neonate transmission of ARGs, to improve infection prevention control measures in LMICs and study the development of the gut microbiome and resistome. We chose β-lactamase genes as markers given the WHO recommendations of first-and second-line treatments for neonatal BS: ampicillin and ceftazidime, respectively. We acknowledge that there are many ESBL and carbapenemase genes; our selection was based on clinical importance and epidemiology. We had limitations with the retrospective recovery of E. coli, K. pneumoniae and E. cloacae for WGS due to loss of the carbapenemase gene and this may be due in part to freeze-thaw effects resulting in plasmid loss. Antibiotic susceptibility testing was performed on a proportion of recoverable isolates. One colony per phenotypically similar bacterial colony was selected for confirmation of the presence of ARGs. We acknowledge the limitation of a microbial culture-based approach that may not have detected the presence of multiple strains of the same species from a single sample. We did not perform a longitudinal study collecting samples across different time points to assess acquisition/ loss of ARGs during the present study, or collect history of antibiotic exposure to understand the effects of antibiotic treatment on the neonatal microbiota. It should be noted that approximately 40% of neonatal samples discussed in the present study originate from Bangladesh (1,117/2,931, Fig. 1) and a limitation of the present study is the under-representation of available neonatal samples from other LMICs. The sociodemographic data collected and used for statistical analysis was largely self-reported and we acknowledge that this approach may have been subject to social desirability and recall bias.
In summary, the present study highlighted the prevalence of the carriage of important β-lactamase genes among the microbiota of mothers and their neonates with either suspected or confirmed sepsis in LMICs. We demonstrated the presence of ARGs in the gut microbiota from the first hours of life that has seldom been reported. We showed that poorer WASH indicators, use of antibiotics and previous infection were probably associated with gut microbiota carriage of bla CTX-M-15 , bla NDM or bla OXA-48 -like genes. Furthermore, the carriage of these genes was most probably associated with neonatal sepsis and adverse birth outcomes. By performing WGS on E. coli, K. pneumoniae and E. cloacae complex isolates, we unveiled the major lineages present in the guts of mothers and neonates in LMIC sites and their AMR-related genetic machinery. WGS showed relatedness between isolates from mothers' and neonates' microbiota and between gut microbiota and those isolates causing neonatal sepsis, warranting future studies. These results contribute to AMR surveillance in previously unexplored settings and populations to

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inform national action plans on better infection prevention practices and to reduce the burden of AMRs in LMICs.

Settings, ethics, participants and study design.
In the present study, the term 'neonates' is used to include all neonates and infants (aged  Table 2). The site abbreviation names were commonly used throughout this publication; however, the country name was used when the results were applicable to all sites within that country.
From November 2015 to November 2017, women in labour (preferably) or immediately post partum were recruited prospectively following their consent and their neonate(s) followed up for the first 60 d of life or until study withdrawal/neonatal death. For neonates lost to follow-up, the information available at the last follow-up point was considered. In addition, neonates who presented to clinical sites with clinically suspected sepsis in the first 60 d of life were recruited (with their mothers) on consent and followed up as described. Demographic and clinical data were collected on pretested study forms by trained researchers. The definitions for clinically suspected sepsis are detailed in https://www.ineosoxford.ox.ac.uk/research/barnards. BS was assigned to neonates with blood culture-positive sample(s), as described elsewhere 10 .
Further details of the study design and sociodemographic and clinical characteristics of mothers and neonates are described elsewhere 41 According to the established protocol, rectal samples were to be taken from all mothers on recruitment and from neonates aged ≥7 d up to 60 d with clinically suspected sepsis. However, during the course of the present study, rectal samples were taken from neonates with clinically suspected sepsis from 0 d of life onward and these samples were also characterized and included in the present study. For this, sterile swabs in Amies Transport Medium with charcoal (Liofilchem) were used as described in https://www.ineosoxford.ox.ac.uk/research/barnards. Swabs were maintained at 4 °C until transfer to Cardiff University (CU) under UN3733 regulations at room temperature.
Ethics approval and consent to participate. Ethical approval was obtained at each of the seven participating countries (Supplementary Table 2 Table 2. In local languages, research nurses provided mothers with study information and collected consent for mother and/or neonatal enrolment. Informed consent was obtained in writing unless this was not possible (due to literacy barriers) and oral consent was collected from the mothers by trained researchers. Oral consent was documented by the participant signing/marking the consent form. Confounder variables Part A: education status of mother; household income equal to or greater than country average; residence water supply; access to soap; frequency of solid waste collection; access to wastewater network; type of residence. Confounder variables Part B: age of mother; maternal visit to hospital in the 12 months before enrolment; maternal attendance to private healthcare in the 3 months before enrolment; maternal visit to traditional healer in the 3 months before enrolment; household income equal to or greater than country average; education status of mother; type of toilet in residence; mothers' handwashing frequency; residence access to wastewater network; frequency of solid waste collection; residence water supply; mother immunocompromised; mother with diabetes.
Confounder variables Part C: mother immunocompromised; mother with diabetes; maternal visit to hospital in the 12 months before enrolment; maternal attendance to private healthcare in the 3 months before enrolment; maternal visit to traditional healer in the 3 months before enrolment; household income equal to or greater than country average; education status of mother. Analyses were performed to understand: Part A: association between the mother's handwashing frequency (explanatory variable) and maternal carriage of ARGs (outcome) and controlled for the variables described in the tabl legend; Part B: association between maternal infection in the 3 months before enrolment in the study (explanatory variable) and maternal carriage of ARGs (outcome), and controlled for the variables shown in the table legend; and Part C: association between maternal usage of antibiotics in the 3 months before enrolment in the study (explanatory variable) and maternal carriage of ARGs (outcome), and controlling for the variables depicted in the table legend. The z-tests were used from multivariable logistic regression models and statistical tests were two sided. For all models, UV = univariate analysis (all data in Supplementary

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Gut microbiota characterization. On arrival to CU, rectal swabs were stored at 4 °C until processing. Mothers' rectal samples were processed on a ratio of a minimum of 1:3 BS:NoBS-related sample per site 10 and all neonatal rectal swabs were processed. Swabs were streaked on three chromogenic agar medium plates (Liofilchem) supplemented with either vancomycin (10 mg l −1 ), vancomycin and cefotaxime (VC, 10 mg l −1 and 1 mg l −1 , respectively) and vancomycin and ertapenem (VE, 10 mg l −1 and 2 mg l −1 , respectively) to select for cefotaxime-resistant GNBs (indicative of the presence of ESBL producers) and ertapenem-resistant GNBs (indicative of the presence of carbapenemase producers). The GNB microbiota grown on VC and VE plates was scrutinized for the presence of bla CTX-M-15 and of bla NDM , bla KPC and bla OXA-48 -like genes, correspondingly, by PCR/multiplex-PCR using the Illustra PuReTaq Ready-To-Go PCR Beads (GE Healthcare) in a Gene Touch Thermal Cycler (Hangzhou Bioer Technology Co., Ltd). PCR conditions, primers (Eurofins) and control strains are described in Supplementary Table 3. Amplicons were subjected to electrophoresis in a 1% agarose (Sigma-Aldrich) gel at 300 V for 35 min in 1× Tris/borate/EDTA buffer containing 25 µl of ethidium bromide. All bacterial cultures were preserved in TS/72 beads (Technical Service Consultants) at −80 °C.
Phenotypically distinct bacterial colonies in VE plates from multiplex-PCR-positive samples were selected and pure cultures obtained by repeated isolation of individual colonies in the same medium. All isolates were subjected to multiplex-PCR and those with a positive result for any of the carbapenemase genes in the study were identified by MALDI-TOF MS (Bruker Daltonik GmbH) and preserved as mentioned before until further analysis. The workflow for sample collection and processing is shown in Extended Data Fig. 8. Due to the high prevalence of bla CTX-M-15 , we did not scrutinize samples for bla CTX-M-15 -positive isolates.
Indian samples were processed locally using the same methodology, except for bacterial isolate identification, which was done using Enterosystem 18R (Liofilchem) and the VITEK 2 Compact Automated System.
BSyn sample results were included in both BS and NoBS groups, because the same mother had neonates with different BS statuses. Hence, the results for each of the 68 samples were accounted for twice. AMR profiles. Antibiotic susceptibility testing was performed using the disk diffusion method for a subset of isolates (n = 584) according to EUCAST v.9 guidelines (2019) 31 , using appropriate control strains to test quality control.  Table 4 shows the antibiotics tested and the disk concentrations, control strains used and EUCAST v.9 breakpoint tables used for interpretation of results.
Genomic analysis of E. coli, E. cloacae and K. pneumoniae. K. pneumoniae, E. coli and E. cloacae isolates from rectal swabs from Bangladesh, Pakistan and Nigeria were selected for further characterization by WGS and bioinformatics analysis. Genomic (g)DNA extraction and Illumina WGS were performed as described 10 .
Briefly, gDNA was extracted using the QIAmp DNA mini-kit (QIAGEN), with an additional RNase step, on the automated QIAcube platform (QIAGEN), and was quantified using the Qubit fluorometer 3.0. Genomic libraries were prepared using Nextera XT v.2 (Illumina), with a bead-based normalization, following the manufacturers' guidelines. A total of 48 isolates is multiplexed per sequencing run to provide a depth of coverage >15×. Paired-end WGS was performed on an Illumina MiSeq using the v.3 chemistry to generate fragment lengths up to 300 bp (600 cycles). For Oxford Nanopore Technology (ONT) sequencing, fresh gDNA was extracted as described above, concentrated using SPRI beads (Mag-Bind TotalPure, Omega) and libraries were generated using the 96-Rapid Barcoding Kit (SQK-RBK110.96; ONT). Sequencing was performed using MinION flow cells (R9.4 and R.10) for a running time of 72 h within MinKnow.
SNP analysis was performed on ST-specific clades using snippy (v.4.6.0) 58 (input paired-end fastq) with BWA and freebayes mapping the reads and calling variants. To maximize SNP calling, for each clade, a high-quality reference was generated 59 using long reads (ONT bioinformatics, see below; Supplementary Table  5 summarizes the genome metrics for each SNP reference genome). Snippy-core was used to concatenate SNPs and snp-sites 60 was used to extract SNPs. Gubbins (v.2.3.4) 61 was used to identify and remove recombination. IQ-tree (v.2.0) was used to generate a maximum likelihood SNP tree 62 . Snp-dists was used to generate a pairwise SNP matrix 63 . SNP trees were outgroup rooted where possible, or mid-point rooted and visualized with iTOL (v.4) 57 .
Illumina paired-end sequence reads were submitted to the European Nucleotide Archive (ENA) and given accession no. PRJEB39293. Hybrid genomes (Illumina and ONT) were submitted to the NCBI and given BioProject accession no. PRJNA767644.
Global isolates for contextual analysis. Approximately 100 isolates of E. coli, K. pneumoniae, and E. cloacae were included in phylogenetic analyses. Isolates were chosen from two searches. First, a literature search ascertained the availability of whole genomes from studies focusing on neonatal studies and/or rectal or intestinal carriage of ESBL/carbapenemase, primarily, but not exclusively in LMICs. Available clinical and other associated data, including country, sample, source and date, were collected, where available.
Second, to provide further context from additional countries and sources, including animal and environmental, between 50 and 80 genomes were chosen from the NCBI Assembly collection. On 5 March 2020 sequence data in fasta file format were downloaded from the NCBI's Assembly resource. For E. coli, 18,761 genomes were downloaded and 18,673 were further identified as E. coli using in-house bioinformatics analysis as described above. A total of 1,790 different STs were found within the E. coli collection. For K. pneumoniae, 8,663 genomes were downloaded and 8,660 were further identified as K. pneumoniae using in-house bioinformatics analysis. A total of 930 different STs were found within the K. pneumoniae collection. For E. cloacae complex, 1,960 genomes were downloaded and 1,886 were further identified as being E. cloacae complex, of which 398 different STs were found.
ABRicate v.0.9.7 was used to screen all genomes for ARGs. To assist choosing both bla NDM/OXA-48 -like-positive and -negative isolates, the dataset was divided according to the ARG output. Genomes were then chosen at random and the accession nos. used to obtain biosample information, including, where possible, country, sample, source and date of isolate.

Statistical analysis.
A formal sample size was not calculated. Sites were asked to recruit all eligible mothers into the overarching study over a period of at least 12 months. All BRs were processed and MRs were processed at a ratio previously described.
Logistic regression models were fitted to maternal and living environment variables (maternal carriage) and healthcare settings, maternal and living environment variables (neonate carriage) to investigate associations with MR and BR β-lactamase gene carriage (bla CTX-M-15 , bla NDM and bla OXA-48 -like genes separately). These were also done for the neonate birth cohort only. Several multivariable analyses were also carried out to explore the association between these variables and maternal/infant carriage of ARGs. Explanatory variables were selected for inclusion in multivariable models on the basis of expert opinion and literature 67 . The variables included are detailed in Supplementary Methods.
We investigated the association between MR/BR β-lactamase gene carriage and neonatal BS by fitting logistic regression models.
To investigate the association between MR β-lactamase gene carriage and birth outcomes (delivery type, timing of birth, perinatal asphyxia, breech presentation and PPROM), multinomial (delivery type (SVD as the base outcome), timing of birth (on time as the base outcome), perinatal asphyxia (no as the base outcome)) and logistic regression (breech presentation and PPROM) models were fitted with birth outcomes as the outcome, and MR β-lactamase gene carriage as the explanatory variable. Conversely, to investigate the association between birth Articles Nature Microbiology outcomes and BR β-lactamase gene carriage, logistic regression models were fitted with BR β-lactamase gene carriage as the outcome and birth outcomes as the explanatory variable.
All models were adjusted for site as a fixed effect. For the association between β-lactamase gene carriage and neonatal BS, we also reported the associations without adjusting for site. Logistic regression models are reported as odds ratios (ORs), 95% confidence intervals (CIs) and P values. Multinomial logistic regression models are reported as relative risk ratios, 95% CIs and P values. P values were adjusted for multiple testing using the Holm-Bonferroni method 68 on a per outcome/model basis (for example, associations with bla CTX-M-15 in MRs were adjusted for separately to associations with bla NDM in MRs, and so on), with a familywise error rate (FWER) of 0.05. Furthermore, owing to the small percentage of missing data, no imputation of missing variables was performed. Given the large number of hypothesis tests reported in the present study, findings where P adj < 0.05 are highlighted in the main text. However, findings from all analyses can be found in Table 1 and the accompanying data, and Supplementary Methods. All analyses used the z-test from a logistic regression model and all statistical tests were two sided. Statistical analyses were conducted using Stata v.16.1.
Extended Data Fig. 7 was edited to reflect significant values with coloured dots using Adobe Illustrator v.25.0.1.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Code availability
Programs were used with default parameters unless specified. The code used is available upon request.